Immunoassay is a powerful technique extensively used in clinical research and medical diagnosis for diseases biomarker screening. Its remarkable specificity and sensitivity is due to the molecular recognition between an antibodies and its target among a huge range of material in a sample. Most of assays are heterogeneous: the immunologic complex is created onto a solid surface, typically the bottom of micro-titer plate, and unbound molecules are removed by several washing step before detection. However, this format presents several drawbacks in which the low capture area and surface-to-volume ratio are the most crucial because they are directly related to the immunoassay sensitivity.
The apparition of magnetic micro-particles as solid support to perform biomarker capture enables to overcome one of those issues. Micrometric beads show a large specific surface with an important binding capacity giving a better capture efficiency. Moreover, thanks to their superparamagnetic property, they are easy to handle what makes the numerous immunoassay separation and washing steps faster to perform. The coupling of the large capture area with the increase in reaction time due to the microfluidic format enables to achieve point-of-care platform detecting biomarker in few minutes with a good sensitivity.
Immuno-agglutination is one of the simplest and fastest one-step immunoassays. It consists in creating aggregates using functionalized beads in the presence of a target with at least two binding sites [15]. This simple procedure is widely applied to immunodiagnostics but it still limited by the slow kinetics of aggregates formation. Several strategies based on microfluidics have shown the possibility to reduce reaction time and improve sensitivity but the procedures remain laborious [16].
Currently, most of beads-based ELISA performed in microfluidic are carried out in continuous flow but for handling and pumping reasons the liquid volumes cannot be lower than few microliters. Reducing the volume of immunoassays is required for some clinical application where the volume sample is available in really small quantity such as new born blood samples. Also, reducing sample or reagent volume is critical for applications in which a multiplicity of criteria or materials or samples have to be screened, as the case in drug screening, high throughput screening, combinatorial chemistry, systems biology, digital biology, synthetic biology, and the like. The screening of neonatal diseases is already developed in most of countries (Clague & Thomas, 2002) [5] but sub-microliter reactions on those samples could give the opportunity to perform multiple analyses from the same sample volume taken. The droplet microfluidic enables to handle small self-enclosed volumes. However, multistep reactions are not easy to execute with usual droplet manipulations such as merging and splitting that is why magnetic droplet handling offers an easy way to overcome those issues and to perform bead-based assays on digital microfluidic platform.
Several methods have been used to manipulate magnetic particles in order to do bio-analytical in an integrated system. First, Shikida et al. (Shikida et al., 2006a) (Shikida et al., 2006b) [6] and [7] introduced beads transfer between two water droplets dispersed in oil in a microfluidic system. The extraction is performed by splitting a small droplet containing particles thanks to a moving magnet and gating structures to retain the drop during extraction. The drop volume had been reduced from about 40 to few microliters in order to perform PCR amplification (Tsuchiya et al., 2008) [3]. Based on this work, many examples of magnetic beads manipulation was demonstrated on digital microfluidic. Most of them are 2D platform which can be divided in two groups: the one where beads are extracted from drops deposed onto a surface with a mobile magnet (Zhang et al., 2010) [8] (Long et al., 2009) [9] and the one based on electro-wetting device in which droplets are moved close to a fixed magnet (Sista et al., 2008) [1]. Although all these systems are able to perform multiple step assays, they are not simple and easy-to-operate mostly due to the droplet creation and positioning onto the surface and/or the magnet movement to implement. Lehmann et al. (Lehmann et al., 2006) [10] reported a platform integrating an array of coils on a PCB substrate coupled with a hydrophobic/hydrophilic surface patterning to split drops. Despite of this integration, the system complexity is highly increased and drop volumes and beads amount are still in the microliter and hundreds of micrograms range.
Biphasic microfluidics offer unique capabilities for the development of flexible and high throughput analytical systems. Platforms combining droplet microfluidics with magnetic particles provide the advantages of heterogeneous assays, while enabling complex operations such as on-chip transport without dispersion, mixing and merging of aliquots. Interesting applications based on this strategy were proposed, using in particular electrowetting on 2D supports [1], but they are relatively low-throughput, and electrowetting raises delicate contamination issues limiting bioassays performances.
It is thus an object of the invention to overcome the above limitations. In particular, in one of its aspects, the invention relates to a microcapillary or microchannel based platform with integrated magnetic tweezers enabling the fast and robust implementation of complex bead-based bioassays in liquid droplets. This approach thus enables us to split, transport, mix and merge droplets reliably while providing high throughput analysis and multiplexing capabilities that are necessary to perform bioassay.
Further, the methods disclosed e.g. in Gu et al., Anal chem, 2011, dx.doi.org/10.1021/ac201678g, may not provide the separation of superparamagnetic particles, without combining said superparamagnetic particles with ferromagnetic particles.
The latter particles, however, do not separate easily after having been magnetized, so this approach of the prior art did not allow resuspension, and strongly limited the number of steps of protocols. These disadvantages might be shared by ferrimagnetic particles, or by antiferromagnetic ones.
In addition, prior droplet systems, may not provide the possibility to perform washing and elution steps.
In other known methods [12], droplets were split by hydrodynamic means, and a magnet was used to move magnetic particles preferentially in one of the daughter droplets. However, in this case the droplet containing the particles and the other have a comparable size, so the separation power is poor.
It is another object of the invention to increase aggregate formation kinetics, reduce volumes and provide a fully automated and low cost platform for immuno-agglutination.
It is an object of the invention, to provide a method for performing chemical, biological, physical or biochemical processes, analysis or reactions, thanks to an improved extraction process.
It is an object of the invention to provide improved methods for performing chemical, biological, physical or biochemical processes, analysis or reactions, wherein superparamagnetic particles are contained in droplets contained in a microchannel.
It is an object of the invention to provide a system that allows to stop magnetic particles contained in a droplet, into a secondary droplet containing a minimal volume of fluid, without stopping the remainder of the droplet.
It is an object of the invention to provide systems for performing various types of assays, optionally comprising several steps, in microfluidic devices that may not comprise microelectrode arrays or microfabricated microcoils.
It is an object of the invention to provide systems for performing various types of assays, optionally comprising several steps, in microfluidic devices wherein droplets are transported by flow or by a pressure difference.